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Bone tissue engineering: Scaffold preparation using chitosan and other biomaterials with different design and fabrication techniques
Accepted Manuscript Bone tissue engineering: Scaffold preparation using chitosan and other biomaterials with different design and fabrication techniques
S. Preethi Soundarya, A. Haritha Menon, S. Viji Chandran, N. Selvamurugan PII: DOI: Reference:
S0141-8130(18)33716-4 doi:10.1016/j.ijbiomac.2018.08.056 BIOMAC 10300
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19 July 2018 7 August 2018 10 August 2018
Please cite this article as: S. Preethi Soundarya, A. Haritha Menon, S. Viji Chandran, N. Selvamurugan , Bone tissue engineering: Scaffold preparation using chitosan and other biomaterials with different design and fabrication techniques. Biomac (2018), doi:10.1016/j.ijbiomac.2018.08.056
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ACCEPTED MANUSCRIPT Bone tissue engineering: Scaffold preparation using chitosan and other biomaterials with different design and fabrication techniques
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S. Preethi Soundarya , A. Haritha Menon , S. Viji Chandran+ and N. Selvamurugan*
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Department of Biotechnology, School of Bioengineering, SRM Institute of Science and
These authors contributed equally to the paper
To whom correspondence should be made:
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Technology, Kattankulathur 603 203, Tamil Nadu, India
Professor
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N. Selvamurugan, Ph.D.
Department of Biotechnology School of Bioengineering SRM Institute of Science and Technology Kattankulathur 603 203. Tamil Nadu India. Cell: 91-9940632335 Email: [email protected] [email protected] 1
ACCEPTED MANUSCRIPT Abstract In the recent years, a paradigm shift is taking place where metallic/synthetic implants and tissue grafts are being replaced by tissue engineering approach. A well designed threedimensional scaffold is one of the fundamental tools to guide tissue formation in vitro and in vivo. Bone is a highly dynamic and an integrative tissue, and thus enormous efforts have been
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invested in bone tissue engineering to design a highly porous scaffold which plays a critical role
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in guiding bone growth and regeneration. Numerous techniques have been developed to
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fabricate highly interconnected, porous scaffold for bone tissue engineering applications with the help of biomolecules such as chitosan, collagen, gelatin, silk, etc. We aim, in this review, to provide an overview of different types of fabrication techniques for scaffold preparation in bone
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tissue engineering using biological macromolecules.
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Keywords: Fabrication, scaffold, bone tissue engineering, chitosan, Runx2
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ACCEPTED MANUSCRIPT Abbreviations Alg - Alginate ALP - Alkaline phosphatase BTE – Bone tissue engineering
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CI - Chitin
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Col - Collagen CMC – Carboxymethyl cellulose
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CS - Chitosan
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FDM - Fused deposited modeling Gel - Gelatin
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HA - Hyaluronic Acid
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HAp – Hydroxyapatite
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LOM - Laminated object manufacturing MSC – Mesenchymal stem cell
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OPN – Osteopontin
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PCL - Polycaprolactone
PEG – Polyethylene glycol PGA - Polyglycolic acid PLGA - Poly-dl-lactic-co-glycolic acid PLLA - Poly-l-lactic acid PVA – Polyvinyl alcohol
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ACCEPTED MANUSCRIPT PS - Polystyrene PVP - Polyvinylpyrrolidone
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Runx2 - Runt-related transcription factor 2
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ACCEPTED MANUSCRIPT Contents 1. Bone-----------------------------------------------------------------------------2. Bone defects---------------------------------------------------------------------3. Bone tissue engineering--------------------------------------------------------4. Biomaterials in bone tissue engineering -------------------------------------4.1 Natural Polymers-------------------------------------------------------
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4.1.1 Chitin/Chitosan ---------------------------------------------
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4.1.2 Collagen/Gelatin---------------------------------------------
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4.1.3 Alginate ------------------------------------------------------4.1.4 Hyaluronic Acid ---------------------------------------------
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4.1.5 Silk ------------------------------------------------------------4.2 Synthetic Polymers------------------------------------------------------
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4.3 Ceramics ---------------------------------------------------------------5. Design of natural composites for bone regeneration ---------------------
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5.1 Biocompatibility property-----------------------------------------------5.2 Biodegradability ----------------------------------------------------------
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5.3 Scaffold architecture ----------------------------------------------------5.4 Mechanical property -----------------------------------------------------
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5.5 Fabrication techniques---------------------------------------------------5.5.1 Freeze drying --------------------------------------------------------
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5.5.2 Solvent casting -----------------------------------------------------5.5.3 Particulate leaching -------------------------------------------------
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5.5.4 Gas foaming --------------------------------------------------------5.5.5 Thermally induced phase separation----------------------------5.5.6 Sol-Gel method----------------------------------------------------5.5.7 Electrospinning ----------------------------------------------------5.5.8 3D-printing ---------------------------------------------------------6. Conclusion----------------------------------------------------------------------7. Future perspective----------------------------------------------------------------
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ACCEPTED MANUSCRIPT 1. Bone Bone is a large hard connective tissue that supports and protects various internal organs and provides structural integrity to the body [1-3]. It undergoes continuous remodeling during the lifetime of an individual and acts as the calcium reservoir of the body [4, 5]. Bone is constituted by collagen which primarily forms the organic part of the bone and calcium phosphate (apatite) crystals that are found as embodiments within the collagen matrix and form
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the inorganic part of the bone [4,6]. Bone mainly comprises of four types of cells namely
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osteoblasts, osteoclasts, osteocytes, and bone lining cells and they dynamically regulate bone
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homeostasis. Bone remodeling is a function of bone formation performed by the osteoblasts, and resorption by osteoclasts and equilibrium between these two functions is important for healing,
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bone regeneration and to maintain the structural integrity of the tissue [7-9]. In mammals, most bone develops through endochondral ossification. In the remodelling process, mesenchymal
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stem cells (MSCs) first differentiate into hypertrophic cartilage followed by differentiation of MSCs into osteoblasts in the surrounding perichondrium that form the initial bone matrix
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followed by vascularization. As this process continues, osteoclasts invade and erode the cartilage to replace the chondrocytes with developed bone tissue [10-12]. The osteoblasts continue to lay
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down the bone matrix to form the mature bone tissue, and while doing so, some osteoblasts deposit onto the matrix, and these form the mature bone cells called osteocytes.
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2. Bone defects
Loss of bone tissue can occur as a consequence of trauma due to accidents, cancer or
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bone defects such as osteoporosis, osteogenesis imperfecta and congenital pseudarthrosis [1214]. In case of long-bone injuries, a significant loss in bone tissue results when the natural
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healing potential of the bone falls far below the required regeneration. Hormonal imbalance caused as a result of aging is also a significant cause of bone loss. Studies showed that the hormone estrogen suppresses osteoclast activation and parathyroid hormone (PTH) regulates bone remodeling [15-18]. Diabetes is yet another major disorder that contributes sizably to the lossof bone tissue. This occurs as a result of elevated levels of free radicals in the surrounding tissues causing an acidic environment, thereby activating the osteoclasts that resorb the bone matrix [19-22]. Allografts and auto-grafts are being employed for replacement of lost bone tissue. However, these options pose major disadvantages such as lack of donors and immunogenic rejection of the graft by the host’s body [23, 24]. To overcome these 6
ACCEPTED MANUSCRIPT disadvantages, bone tissue engineering (BTE) is now being employed in the treatment of bone defect/loss. 3. Bone tissue engineering Creating tissue constructs that mimic the bone in both structure and function has been a challenge so far. A bone graft or scaffold should mimic the structure and properties of the natural bone extracellular matrix (ECM) and provides all the necessary environmental cues
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found in natural bone. This is where BTE comes into play, and the current trends of regenerative
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medicine have focused on the creation of 3D-scaffold with the help of biomaterials and cells that
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can mimic the ECM, support the formation of new tissue/bone, and meanwhile, degrade as new bone is produced [25]. Various technologies come together to construct porous scaffold to
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regenerate tissues/organs and also for the controlled and targeted release of bioactive agents in tissue engineering applications. The optimal characteristics of a scaffold are the strength, the rate
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of degradation, porosity, and microstructure, as well as their shapes and sizes of the polymeric scaffold [26, 27].
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The following are some of the features that need to be possessed by the scaffold to be used in BTE efficiently. (i) The scaffold biomaterial should possess flexibility, i.e. it should be
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easy to fabricate the material into 3D-structures. (ii) The topography of the biomaterial fabricated into 3D-dimensional structures should promote cell adhesion, cell infiltration, and
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growth. This means that these 3D-structures should possess well interconnected pores that allow cell infiltration and nutrient exchange. Any biomaterial that supports attachment of cells and
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growth of bone on its surface is considered to be osteoconductive. (iii) The rate of degradation of the 3D-structured biomaterial is also a very crucial characteristic. The biomaterial must undergo
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resorption as new bone is formed, i.e. the rate of formation of the new bone tissue should be proportional to the rate of degradation of the biomaterial, and its degradation byproducts must not cause inflammation or toxicity in surrounding tissues or the whole system of the host. (iv) Presence of concavities or spaces between individual particles of the biocomposite scaffold, which aid in heterotopic bone formation without being disturbed by high circulation body fluid or mechanical forces due to implant movement [12, 28-30]. 4. Biomaterials in bone tissue engineering Numerous scaffolds are created from a diverse range of biomaterials and were produced using a plethora of fabrication techniques in attempts to regenerate bone in the body. Regardless 7
ACCEPTED MANUSCRIPT of the site of implantation, a number of key factors are to be taken into consideration when designing or determining the suitability of a scaffold for use in tissue engineering. Biomaterials play a crucial role in the field of BTE by acting as artificial frameworks namely scaffolds, matrices, or constructs [31]. Three distinct groups of biomaterials namely natural, synthetic polymers and ceramics are used in the fabrication of scaffold for tissue engineering. Polymers include natural and synthetic polymers [32].
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4.1 Natural Polymers
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Naturally derived polymer such as chitin (CI), chitosan (CS), collagen (Col), gelatin
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(Gel), alginate (Alg), hyaluronic acid (HA) and its composites are resourceful biomaterials that play a predominant role in BTE.
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4.1.1 Chitin/Chitosan
CI is the most abundant natural amino polysaccharide and it is mainly extracted from
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crustaceans (shrimps, crabs, lobsters, etc). CI and CS are considered as a versatile biomaterial. Although CI possesses various industrial applications, its usage is limited in BTE due to its weak
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solubility. On the other hand, the deacetylated CI derivative-CS is a well-established natural polymer. It mainly comprises of units namely (1-4) glycosidic bonds linked d-glucoasmine
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residues with a variable number of randomly located N-acetyl-d-glucosamine (NAG) groups. CS possesses structural resemblance with glycosaminoglycans (GAG), one of the components of
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ECM that interacts with collagen fibers and plays a vital role in cell-cell adhesion. CS has been given great importance in the field of scaffold fabrication as it contains various properties such
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as biocompatible, biodegradable, anti-bacterial effect and many more [31-33]. To enhance the properties of CS such as mechanical strength and structural integrity for BTE applications, other
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natural/synthetic polymers, metals and ceramics are added to CS, and used as 3D-lyophilized scaffolds, hydrogels/film, and electrospun mats. In many studies, polymers such as Alg, Gel, PCL and bioactive nano ceramics such as HAp, SiO2, TiO2, ZrO2, etc have been used to increase the mechanical strength of CS biocomposites [4, 31-39]. 4.1.2 Collagen/Gelatin Col is the most abundant and widely distributed protein present in our body. Col types 1, 2, 3, 5 and 9 are known to form fibres out of the 29 different types of collagen that have been identified so far. Structurally, Col is composed of 25 different alpha chains with each alpha chain composed of repeating units of Gly-X-Y. The X and Y residues may be proline or 48
ACCEPTED MANUSCRIPT hydroxyproline. The fibres are the final quaternary structure of the Col protein [40]. In BTE, Col obtained from various sources such as marine sponge and ruminants is widely used in combination with a variety of other materials for fabrication of electrospun fibres, scaffold matrices and hydrogels to aid in targeted delivery, stem cell differentiation and osteogenesis [ 41, 42]. Recombinant human-like Col is being synthesized in large scale using bacteria and yeast and aided in higher fibroblast compatibility and increased bone growth [43, 44]. The properties
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of Col such as high biocompatibility, matrix mimicking and biodegradation make it a suitable
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candidate for BTE. Col is highly biodegradable and may degrade off quite quickly when
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implanted in the target site. Col-based scaffolds served as the best carrier for targeted delivery small molecules of antagonists of miR-16, which inhibits Runx2 mRNA (a bone transcription
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factor), thereby promoting osteogenesis [45]. However, Col being a protein is difficult to be fabricated by certain fabrication techniques that involve heat and cross-linking without altering
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its native structure [40]. Gel is a product of collagen denaturation and has the RGD (arginine, glycine, and aspartate) binding sequence that improves cell adhesion. It has been widely used
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separately and as a mixture with many natural polymers including Col, proteins such as fibroin, ceramics such as apatite, and other synthetic and semi synthetic polymers [46-48].
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4.1.3 Alginate
Alg is a natural anionic polymer and it is mainly extracted from brown seaweed. It is
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composed of guluronic acid and mannuronic acid, and possesses properties such as less toxicity, high abundance resources, low cost, gel forming property, biocompatibility and has the potential
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to mimic ECMs. Alg composites are prepared by various cross-linking methods, and it is used in wound healing, tissue engineering and delivery/carriers of drug and other bioactive agents in
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biomedical applications. Several Alg-based biocomposite scaffolds have been reported to possess bone regeneration property. Amongst which Alg/CS composites are the most studied composites that is said to regenerate bone tissue. HAp was blended with Alg in co precipitation method to increase the mechanical strength and these scaffolds have also been reported to possess excellent osteogenic effect [49, 50]. Alg was blended with many other polymers/ceramics such as Col, Gel, nano bioactive glass to increase its mechanical strength, adhesive property and osteogenic differentiation ability [50-53].
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ACCEPTED MANUSCRIPT 4.1.4 Hyaluronic Acid HA is a polysaccharide mainly composed of alternating residues of D-glucuronic acid and N-acetyl glucosamine. It is found in almost all the living organisms, mainly in the connective tissues and load bearing joints, etc. HA is highly viscous, elastic, biocompatible, nonimmunogenic and biodegradable nature makes it an excellent polymer of choice for BTE. It has been shown that HA as such or in mixture with other polymers and ceramics promoted bone
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regeneration [54, 55]. HA is mainly used as an aqueous binder, example in HA/HAp/TCP
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scaffolds where it not only increased osteoconduction but improved clinical handling and
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application [56]. In another study, HA/Gel hydrogels were incorporated into biphasic calcium phosphate (BCP) ceramic to obtain a unique micro- and macroporous structure and acted as
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novel bone substitute for BTE [57]. HA-coated scaffolds were also prepared to enhance the bioactivity of scaffolds prepared from synthetic polymers such as PLA, PVP, PLGA, etc [58,
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59]. Therefore, HA offers great potential as a scaffolding material. 4.1.5 Silk
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Silk fibers are produced by silkworms, bees, spiders, mites, and scorpions. They are composed of two different proteins namely silk fibroin (fibrous) and sericin (globular). Silk
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fibroin is structurally composed of heavy chains and light chains. Sericin coats these two chains of fibroin. Silk fibers are known for their elasticity and other strong mechanical properties. Silk
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protein from tasar silk is rich in RGD sequence which makes it a suitable candidate for BTE [60, 61]. MSCs cultured on silk fibers showed an increase in alkaline phosphatase (ALP) activity and
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after 12 days of treatment, they showed increase in mineralization [62]. There are reports indicating that silk fibroin used in combination with other biomaterials showed enhanced bone
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regeneration. Electrosprayed nanoparticles and hydrogels made of silk fibroin and HAp showed increase in cell proliferation, ALP activity, mineralization and osteocalcin expression, thus proving it supports for mature bone formation [63, 64]. Addition of magnetite particles to silk fibroin/ CS scaffolds aided in synthesis of scaffolds with desired pore sizes [65]. Polymers such as carboxyl methyl cellulose in combination with silk fibroin have been used in synthesis of layer-by-layer assembled fibers to form a 3D-scaffold for BTE [66]. Recently, a patented study showed that surface modification of silk fibroin with ethylene glycol increased adhesion of cells to its surface and decreased thrombosis [67]. This application may be employed to enhance the bone regeneration properties of silk fibroin. 10
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polycaprolactone (PCL), polyvinylpyrrolidone (PVP) and poly-dl-lactic-co-glycolic acid (PLGA). These polymers are often cheaper than natural polymers and also have much success as they can be fabricated with a tailored architecture, long shelf life and production in large scale.
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Their degradation characteristics can be controlled by varying the polymer itself or the
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composition of the individual polymer. They also have drawbacks including the risk of rejection
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due to reduced bioactivity [2, 26, 68-70]. 4.3 Ceramics
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Ceramics such as hydroxyapatite (HA), tricalcium phosphate (TCP), bioactive glasses and glass-ceramics have been shown to have osteoconductivity, high compressive strength, and
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good bone integration. However, their applications in tissue engineering have been limited because of their brittleness, difficulty of shaping for implantation and biodegradability. We can
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overcome these issues by merging natural biomolecules with synthetic polymers, ceramics or by using biocomposite materials that improve scaffold properties, improve tissue interaction and
applications [2, 26, 68-70].
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thereby allow controlled degradation and improve the biocompatibility in tissue engineering
5.1 Biocompatibility
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5. Design of natural composites for bone regeneration
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An important criterion of any scaffold for tissue engineering is that it must be biocompatible; the scaffold must promote cell adhesion, vascularization, the supply of nutrients
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and oxygen to the cells and also support normal cellular activity including molecular signaling systems. After implantation, the scaffold or tissue engineered construct should not elicit an undesirable local or systemic responses to the host. Cytotoxicity, genotoxicity, immunogenicity, mutagenicity, and thrombogenicity are some of the undesirable effects to be eliminated as it might cause severe inflammatory response thus reduce healing or cause rejection by the body [25-26, 71-73]. 5.2 Biodegradability The degradation rate of the scaffold must be in designed in such a way that it provides the necessary structural support and also allows the body's cells, over time, to eventually replace 11
ACCEPTED MANUSCRIPT the implanted scaffold or tissue engineered construct. The scaffold must be biodegradable and are not intended as permanent implants. It is related to biocompatibility because the degraded products should also be non-toxic and able to exit the body without interference with other organs [4, 26, 74-75] 5.3 Scaffold architecture Another indispensable requisite for scaffold is its architecture, porosity and pore size.
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The scaffold should have an interconnected structure and high porosity to ensure cellular
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penetration and adequate diffusion of nutrients to cells within the construct. Cells primarily
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interact with scaffold via the chemical groups or ligands on the material surface. Scaffold synthesized from natural extracellular materials possess these ligands in the form of Arg-Gly-
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Asp (RGD) binding sequences, whereas scaffold made from synthetic materials may require deliberate incorporation of these ligands through, for example, protein adsorption. The ligand
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density is influenced by the available surface within a pore of the scaffold to which cells can adhere [26, 74, 76-77].
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Figure 1 5.4 Mechanical property
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Ideally, the mechanical properties of scaffold should be consistent with the anatomical site into which it is to be implanted, and it must also be strong enough to allow surgical handling
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during implantation. Bone supports the body weight, and it possesses very high compressive strength. In general, the compressive modulus ranges for trabecular bone and cortical bone from
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0.01 to 2.0 GPa and from 14 to 18 GPa, respectively. The scaffold should maintain sufficient mechanical properties until newly formed bone can assume a structured role. Fabricating
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scaffold with passable mechanical properties is one of the great challenges in engineering bone or cartilage. For these tissues, the implanted scaffold should have sufficient mechanical integrity to function from the beginning of implantation to the completion of the remodeling process. A further challenge is that healing rates vary with age; for example, in young individuals, fractures normally heal completely back to its same integrity, but in the elderly, the rate of repair slows down. So this parameter should also be considered when designing a scaffold for orthopedic applications. From this, it is evident that a balance between mechanical properties and porous architecture is key to the success of any scaffold [4, 26, 30, 72, 81,82].
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ACCEPTED MANUSCRIPT 5.5 Fabrication techniques The fabrication technique is the final criterion for designing scaffold for tissue engineering applications and all the criteria listed above depend on it. Table 1 5.5.1 Freeze-drying Lyophilization or freeze-drying has evolved as a conventional fabrication technique for
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BTE. This technique has often been used for food products, biological materials, and drug
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delivery systems. The advantage of this technique is to fabricate a scaffold without the use of a
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high temperature or a separate leaching step and can produce a scaffold of various sizes and shape as required. It is a dehydration technique based on the removal of water/solvent from the
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frozen solution by sublimation under vacuum, at low pressure, leading to an anhydrous or almost anhydrous 3D-structure. The method consists of creating an emulsion by homogenization of a
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polymer solution (in an organic solvent) and water mixture, rapidly cooling the emulsion to lock in the liquid state structure, and removing the solvent and water by freeze-drying. Over 90% of
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porosity can be expected from this technique with the pore size ranging from 20 to 200 µm. The pore size is mostly controlled by controlling the parameters such as rate of the freeze, pH,
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polymer concentration and temperature. For this process, vacuum with high power is needed to come out with a scaffold which has high porosity and interconnectivity. Several natural
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biomolecules and synthetic polymers such as silk proteins, CS, Col, cellulose, Gel, PGA, PLLA, PLGA, PLGA/PPF blends, etc. are widely been used in the freeze-drying technique. But this
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technique is a slow and expensive method, since the cycle may be long, necessitating considerable energy consumption. Freeze-drying can be coupled with techniques such as solvent
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casting, particulate leaching, gas foaming, porogen leaching or by emulsion freeze-drying [4, 81,
5.5.2 Solvent casting
It is one of the easiest and inexpensive techniques for fabricating scaffold. It is completely based on the evaporation of the solvent present in the polymeric solution producing porous scaffolds. The scaffold can be prepared by two methods namely (i) The polymer solution is poured into the mold and allowed to rest for the evaporation of the solvent to obtain a porous 3D-scaffold. (ii) The mold is dipped into the polymeric solution and allowed to rest to obtain a polymeric membrane. Some of the solvents used for casting are methylene chloride, methanol, 13
ACCEPTED MANUSCRIPT acetone, dichloromethane, chloroform, etc. The major drawback of this technique is that the solvent used may be toxic and might retain some toxicity thus they are either combined with freeze-drying or particulate leaching technique for better results [129-131]. 5.5.3 Particulate leaching This method involves mixing water-soluble salt (e.g., sodium chloride, sodium citrate), sugar particles or a porogen into a biodegradable polymer solution. The polymeric solution is
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then cast into the mold of the desired shape. After the solvent is removed by lyophilization, the
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salt particles/porogen is leached out to obtain a porous structure. This method is highly
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advantageous because size of the pores can be controlled by salt/polymer ratio and also the size of the particle added. However, the pore shape is limited to the crystal shape of the salt. Another
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drawback is the difficulty in removing soluble salt/particles from the interior of a polymer matrix which makes it hard to fabricate very thick scaffolds. In fact, most of the porous materials
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prepared by solvent casting and particulate leaching method are restricted by thickness [128, 130, 132].
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5.5.4 Gas foaming
Gas foaming process can be used to fabricate highly porous polymer foams without the
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use of any solvents. In this method, an inert gas usually carbon dioxide (CO2) is used as an agent for the formation of polymer foam. Solid polymer disks are exposed to high-pressure gas which
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leads to the nucleation and growth of gas bubbles in the material. They are then freeze-dried to get a 3D-structure with a pore size of around 100 µm and porosity up to 93%. The disadvantage
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of this method is that it mostly yields a closed-pore structure, with less interconnectivity. This can further be combined with other methods to get a better scaffold architecture [128, 133, 134].
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5.5.5 Thermally induced phase separation Thermally induced phase separation technique is based on changes in thermal energy to induce separation of a homogeneous polymer solution into a multi-phase system domain by a quench route. Firstly the polymer is dissolved in a solvent (phenol or naphthalene) at a high temperature, followed by dispersion of biologically active molecule in these solutions. On declining the temperature the separation is induced, the homogeneous solution separates into a polymer-rich phase and solvent-rich phase either by solid-liquid de-mixing or liquid-liquid phase separation mechanism. Then the solvent is removed by extraction, evaporation, and sublimation to give porous scaffold with bioactive molecules integrated into to that structure. The factors that 14
ACCEPTED MANUSCRIPT affect the pore morphology of the scaffold are the type of polymer, solvent, the concentration of the polymer solution and phase separation temperature. The advantage of this technique is that it can easily combine with other fabrication technology and have good mechanical properties to design three-dimensional structures with controlled pore morphology [128, 135]. 5.5.6 Sol-Gel method Simply put, sol-gels are very porous, highly ordered structures analogous to sponges.
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This gel is made up of discrete particles or polymers. Most frequently used precursors are
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chlorides or metal alkoxides. These precursors are hydrolyzed and polycondensed for the
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formation of colloids. Lesser amounts of dopants like organic dyes and also rare earth elements can be used in the sol for homogeneous distribution of the contents present in the final product.
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The major steps involved in the sol-gel process are mixing, casting and gelation. In the stage of mixing, a colloidal solution is formed via mechanical mixing of colloidal particles in water as a
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solvent. The metal alkoxide precursor reacts with water and undergoes hydrolysis and polycondensation reactions. The formation of a colloidal dispersion of extremely small particles
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(1-2 nm) takes place that finally converts into a 3D-network of the corresponding inorganic oxide. In the stage of casting, sol has a low viscosity which makes it easier for it to cast into a
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mold or shape. A suitable mold is chosen to avoid the sticking of the polymeric gel to the mold. During gelation process, 3D-networks start forming from colloidal particles. Gelation results in
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agglomeration with a colloidal particle which is due to interactions among the components which can be electrical. The next steps are the aging and drying of the newly created structure,
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further solidifying the material. It is then followed by dehydration or chemical stabilization of the sol-gel to create the ultrastable, porous material. The final step is densification, tempering
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the gel with high heat to solidify the gel. This process has been extensively used in the field of ceramics, glass, and thin-film coatings. Thin metal oxide films can also be produced using this method. The sol-gel process is preferred due to its economic feasibility and the low-temperature process which gives us control over the composition of the product achieved [136-138]. 5.5.7 Electrospinning Electrospinning technique is a unique approach where the scaffold is prepared using electrostatic forces to produce fine fibers from polymer solutions or melts, and the fibers thus produced have a thickness ranging from nanometer to micrometer and a larger surface area than those obtained from conventional spinning processes. More than 200 different polymers and 15
ACCEPTED MANUSCRIPT composites are used in the electrospinning such as silk fibroin, chitosan, gelatin, collagen, etc. and many synthetic polymers (PCL, PVP, PLLA, etc.) are also used in this technique [130]. A high DC voltage in the range of 10-15 kV is necessary to generate the electrospun fibers, and it is conducted at room temperature with atmospheric conditions. Presently, there are two standard electrospinning setups, vertical and horizontal. Chiefly, an electrospinning set up consists of three major components: a high voltage power supply, a spinneret (e.g., a pipette tip) and a
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grounded collecting plate (usually a metal screen, plate, or rotating mandrel). Most of the
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polymers are dissolved in a solvent to form a homogeneous solution. Then the polymer fluid is
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introduced into the capillary tube and in the presence of a high voltage source, a certain polarity is introduced into the polymer solution or melt, which is then accelerated towards a collector of
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opposite polarity at a constant flow rate [139-140]. However, some polymers may emit an unpleasant or even harmful smell, so the processes should be conducted within chambers having
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a ventilation system [140]. In the electrospinning process, the polymer solution becomes uniformly charged due to the presence of high voltage, and this causes a repulsive force within
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it. When the electric field applied reaches a critical value, the repulsive electrical forces overcome the surface tension forces thereby causing a charged jet of the solution to eject from
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the tip of the Taylor cone and moves towards the opposite polarity or neutral (grounded) charged electrode which is placed nearby that attracts and collects the fibers. The end product is the
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formation of continuous nanofibers. Major advantages of this technique are that it is very versatile, non- invasive and does not require high temperature for the generation of fibers. This
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technique has seen a remarkable increase in research and commercial attention in various fields over the past decade as it can provide structures that are essential for cell growth and subsequent
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tissue organization. With the expansion of this technology, several research groups have developed more sophisticated systems that can fabricate more complex nanofibrous structures in a more controlled and efficient manner [90,91]. These innovations include coaxial electrospinning – the transformation of nanofibers from a single component to complex multilevel structures by multiple feed system through coaxial capillaries, multi-jet, and blow-assisted multi-jet electrospinning technique increased the throughput of the fiber yield and the mutual electrostatic repulsion was reduced in the later technique [141, 142] Many parameters affect the morphology and diameter of electrospun fibers,and some of these important parameters are (i) the type of polymers used, the viscosity of polymer solution, 16
ACCEPTED MANUSCRIPT the surface tension of the solvent in the polymer solution, (ii) the rate of feeding of the polymer solution, the distance between the needle and collector, needle tip size, and (iii) the temperature and humidity of the surrounding environment [143]. Extremely high surface-to-volume ratio, tunable porosity, malleability to conform to a wide variety of sizes and shapes and the ability to control the nanofiber composition are some of the advantages of this technique. The high surface to volume ratio adds benefit as it enables drug loading, cell attachment and infiltration for bone
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growth at the site of implantation [102]. Natural polymers offer good biocompatibility and
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hydrophilicity. However, these polymers often have relatively lesser mechanical strength.
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Therefore, blending synthetic polymers with natural polymers to obtain electrospun fibers overcome these drawbacks alongside decreasing the hydrophobicity of the synthetic constituent
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[144]. Growth factors and phytochemicals have been delivered targeting a specific tissue, and controlled release of these factors could be aided through the blending of different polymers at
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different ratios [139, 145]. 5.5.8 3D-printing
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The traditional methods for fabricating 3D-porous scaffold inBTE applications are such as polyurethane foam templating, solvent casting, melt molding, and freeze-drying; however, it
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is difficult to control the pore interconnection, pore size and overall porosity of the scaffolds. To overcome these, the 3D-printing technique was used to fabricate tissue engineering scaffold
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[120]. In tissue engineering, bioactive cells mixed with hydrogels are deposited layer by layer to form the 3D-structure that mimics the organs [146-150]. These methods create a 3D-scaffold for
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tissue engineering with the help of CAD program like AutoCAD, AutoDesk, etc [151]. 3D-printing, also known as rapid prototyping and additive manufacturing is a technique
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that was first described in the year 1986. This method was initially called stereolithography (SL) by depositing thin layers of a material on top of each other by curing the layers using ultraviolet radiation [152]. Two-photon absorption by photopolymerization for preparation of 3D-organs was derived from this technique where a monomer gel and photo-initiator material were mixed in layers and subjected to the laser. The photo-initiator contains two active photons. On application of the laser, the photons are released and these act as the active species for the monomer to polymerize locally [153, 154]. Similarly, another method was developed namely Selective Laser Sintering (SLS) to make 3D-organs. SLS uses a high power laser to sinter polymer powders to generate a 3D-model [151, 155]. Although it was successful in generating 17
ACCEPTED MANUSCRIPT complex 3D-parts, it had some serious disadvantages. Inventors found that the resolution of printing were direction dependent, i.e. the 3D-parts produced using this method were found to be structurally stronger in one direction but weaker in the other depending on whether the part is parallel to the direction of the laser. The second major disadvantage was the use of high temperatures that caused thermal warpage, and this made the method less suitable to use thermosensitive biological molecules like proteins and cells in the fabrication of 3D-organs [156,
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157].
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Later in the year 2000, the first inkjet-based technique was used for printing proteins and
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endothelial cells into 3D-structures. It was done by modifying the nozzles of a commercial inkjet printer and feeding in software that enabled the user to change the nozzle in the desired x and y
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position. In drug delivery, inkjet printers are most commonly used the technique. Similar to the household inkjet printers, these printing systems deposit small droplets of the drug on liquid
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films that encapsulate the drug droplet to form microparticles which can be used for drug delivery. This method was more advantageous than SLS as no high temperature was employed
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and thermosensitive biomolecules could survive the technique [158, 159]. Powder-based printing utilizes an inkjet printing head that continuously sprays the drug,
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cells or other bioactive molecules along with a binder onto a bed of powdered polymer until a 3D-scaffold is formed. Although this technique aided in the fabrication of scaffold with
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relatively higher mechanical strength, it is not compatible to fabricate 3D-tissue substitutes containing cells or proteins due to the use of high temperature [158, 160, 161]. Many polymers
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such as bentonite, dextrin, maltodextrin, saccharose, edible oils and other resins are used as binders. These binders typically dissolve in the solvent used for bioprinting and upon bringing
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back to room temperature; it hardens and sets to provide mechanical strength to the ceramic/polymer base. Dextrose, maltodextrose,and saccharose are derivatives of sugar and can be easily absorbed as glucose by hydrolysis in our body [161-163]. At very high temperatures, these sugar-based polymers were pyrolyzed to carbon [164, 165]. Polymers such as Teflon (polytetrafluoroethylene) and polypropylene may also be used as binders along with natural or semi-synthetic materials such as collagen as they provide higher mechanical strength to the scaffold and also help in preventing degradation and maintaining the structural integrity of the scaffold while using acidic solvents. However, use of these polymers as binders are disadvantageous as they account for very low porosity and the pore sizes of the scaffold can not 18
ACCEPTED MANUSCRIPT be controlled during synthesis [119]. Acoustic droplet ejection is derived from inkjet printing where an ultrasound pulse ejects low volumes of liquids upwards onto a supporting medium [157, 167-169]. Following these inventions, modifications were done to the existing printers to develop the extrusion-based printing such as Fused deposited modelling (FDM) and Laminated object manufacturing (LOM). Extrusion printing is a modification of inkjet bioprinting where an air
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force plunge or mechanical screw plunger is used to print uninterrupted cylindrical lines rather
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than a single bio-ink droplet [116, 117]. In FDM, a 3D-matrix is fabricated by extruding
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thermoplastic materials and depositing the semi-molten materials onto a stage layer by layer. LOM method uses defined sheets of plastic or metal/plastic composite and stacks them layer by
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layer to form a 3D-scaffold [151, 157, 169]. Extrusion-based printing employs both high and low temperature processes, however at low-temperature, they result in less mechanical strength
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of the scaffolds. FDM is a high-temperature process, and hence only thermoplastic materials are used and are not cytocompatible [169].
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Many 3D-printed polymers and ceramic scaffolds have been tested in vivo for their cytocompatibility and bone regeneration capability. Silicon-substituted HAp/PCL and
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demineralized bone matrix scaffolds prepared using rapid prototyping technique were osteoinductive and showed high osseointegration and aided good bone regeneration in rabbit
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models [170]. Resorbable dicalcium phosphate (DCP) scaffold synthesized using the SLS method was found to enhance bone regeneration in rat models. The synthesis involved the
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conversion of α/β-tricalcium phosphate (Ca3(PO4)2, TCP) powder to brushite (dicalcium phosphate dihydrate) and tricalcium phosphate which upon treatment with phosphoric acid and
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hydrothermal conversion yielded DCP with very high porosity and were not suitable for longterm stable implants [171]. DCP scaffold synthesized by other methods of additive manufacturing such as vat polymerization, powder bed fusion, material extrusion, and binder jetting also showed osteogenic potential in vitro but needs to be validated using in vivo studies [172]. 3D-printed calcium silicate scaffolds, collagen scaffold loaded with DNA complexes and composite calcium phosphate/collagen scaffold implanted into femur defects and PCL/PLGA blended scaffold(using FDM) in the calvarial defects in rats resulted in higher bone regeneration compared to tricalcium phosphate scaffold [119, 173]. Hydrogels are the most preferred biomaterial for tissue regeneration because of their adjustable mechanical properties and 19
ACCEPTED MANUSCRIPT hydrating capability which help it to mimic the biological tissue itself. Cell-laden hydrogels of PEG, gelatin and fibrin and their derivatives have been successfully used in studies to show bone regeneration. Extrusion method is the most common method used for cell-laden hydrogel synthesis [121, 174, 175]. The cell-based printing uses a syringe pump filled with the solvent that moves in z-axis and the substrate moves in y-axis as shown in Fig. 2A [184]. The solvent pump speed and solvent droplet velocity can be controlled by providing differential voltage as
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shown in Fig. 2B[185]. The higher the voltage, the higher would be the solvent droplet velocity.
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This factor is critical, and the velocity of the solvent droplet is to be maintained as it determines
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the pore size and distribution of the scaffold [176, 177]. PCL and PLGA are synthetic polymers, and hydrogels made from these polymers using rapid prototyping showed very good bone
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regeneration property in rabbit tibias [173]. Strikingly, one study used the human-safe filamentous virus that selectively infects bacteria to form a matrix for bone tissue engineering. In
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this study, the RGD sequence was fused to the phage DNA, resulting in several copies of the phage filaments with highly distributed RGD sequences. This was implanted in sites of radial
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bone defects in rats and showed high osteogenesis and angiogenesis in a period of eight weeks [178]. Currently, studies are being done to optimize materials to make a patient-specific scaffold
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for bone regeneration using the computerized tomography (CT) scan of human subjects to print large bone substitutes. Using open source coding software, the CT scans are extrapolated, and
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3D printed to form the bone replacements that are anatomically similar to the scan with a similar pore size and infill density. The scaffold could then be coated or filled with the stem cells from
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the patient and implanted back into the patient’s body, thereby improving bone regeneration and eliminating any chance of rejection [179-183]. Currently, the 3D-printed polymer scaffolds are
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being tested and studied to use as effective drug delivery systems and tissue replacements in the field of surgery and medicine using natural polymers such as CS. Figure 2
6. Conclusion BTE is regarded as the best alternative approach to the conventional bone grafting techniques due to various factors. Scaffold is the basic subunit that provides mechanical strength, a site for cell attachment, proliferation, and differentiation. Polymer selection and scaffold fabrication techniques are the key factors to achieve these goals. The scaffold can be fabricated using a variety of techniques based on the site of implantation, the nature of polymers and their 20
ACCEPTED MANUSCRIPT characteristics. Natural polymers such as CS, Col etc are proved to be excellent for bone regeneration but an ideal fabrication method for successful implantation is yet to be found. A lot of inventions and developments have been made in the last decade in the field of tissue engineering to fabricate an ideal scaffold. Yet, there have been several drawbacks that need to be addressed. Thus, the fabrication of scaffold with advanced techniques using different types of
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biomaterials will still constitute to be the center of research efforts in the field of BTE.
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7. Future perspective
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As discussed, there is a number of a method or techniques used to fabricate a scaffold. While each technique has its own share of merits and demerits, it is necessary to select a suitable method to satisfy the requirements of the specific type of tissue to be repaired. Since the
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requirements of an ideal scaffold for BTE is complex, proficient knowledge from the different fields of science such as biomedical engineering, material science, chemical engineering, etc. is
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required. Extensive process-property optimization will be required to achieve this goal. Demand for novel fabrication technique will increase in the fore coming years due to their ability to
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design a scaffold that can be tailored for a specific patient and clinical needs.
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printing of bone fractures: a new tangible realistic way for preoperative planning and
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education, Surg. Innov. 22(5) (2015) 548-551.
[181] C. L. Ventola, Medical applications for 3D printing: current and projected uses, Pharm.
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Ther, 39(10) (2014) 704.
[182] M.M. Barak, M. A. Black, A Novel Use of 3D-Printing Demonstrates Structural Effects of
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Osteoporosis on Cancellous Bone Stiffness and Strength, J MechBehav Biomed Mater. 78 (2018) 455-464. [183] J. d ards,
.
ogers, he
cc racy and
pplicability of 3
odeling and Printing
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l nt Force ranial nj ries, J. Forensic Sci. 63(3) (2018) 683-691.
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[184] T. Peng, Analysis of energy utilization in 3d printing processes, Procedia CIRP. 40 (2016) 62-67.
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[185] M. Lee, H.Y. Kim, Toward nanoscale three-dimensional printing: Nanowalls built of
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electrospun nanofibers, Langmuir. 30(5) (2014) 1210-1214.
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ACCEPTED MANUSCRIPT Figure legends Figure 1. Macroscopic and microscopic images of scaffolds fabricated by different methods in bone tissue engineering. (A) - (D) show the macroscopic images of scaffolds prepared by freezedrying, electrospinning, sol-gel method, and 3D-bioprinting, respectively, and (E) - (H) show the corresponding SEM images of the scaffolds. Reprinted with permission from [77]
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Balagangadharan et al., Chitosan/nano-hydroxyapatite/nano-zirconium dioxide scaffolds with
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miR-590-5p for bone regeneration. Int. J. Biol. Macromol. 111(2018) 953-958; [78] S. Surucu
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and H.T. Sasmazel, Development of core-shell coaxially electrospun composite PCL/chitosan scaffolds. Int. J. Biol. Macromol. 92 (2016) 321-328; [79] Pan et al., Preparation and characterization of electrospun PLCL/poloxamer nanofibers and dextran/gelatin hydrogels for
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skin tissue engineering. PLoS One. 9(11) (2014) e112885; [80] Park et al., Fabrication and characterization of 3D-printed bone-like β-tricalcium phosphate/polycaprolactone scaffolds for
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dental tissue engineering. J. Ind. Eng. Chem. 46 (2017) 175-181. Figure 2. A schematic diagram of a 3D-bioprinter. (A) represents the schematic diagram of a
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3D-bioprinter with the substrate in the moveable Y-axis drive. The Z-axis drive is connected
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to a moveable platform, which is connected to a syringe pump containing the solvent or polymer (B). The syringe pump speed and solvent droplet velocity are controlled by
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supplying differential voltage. Reprinted with permission from [184] T. Peng, Analysis of energy utilization in 3d printing processes. Procedia CIRP. 40 (2016) 62-67; [185] M. Lee H.Y.Kim,
Towardnanoscale
three-dimensional
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and
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electrospunnanofibers. Langmuir. 30(5) (2014) 1210-1214.
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printing:
Nanowalls
built
of
ACCEPTED MANUSCRIPT Table 1. In the study of bone regeneration in vitro and in vivo, a number of the following different types of fabrication techniques, biomaterials, bioactive molecules and experimental model system were used.
Fabrication
Materials
Additive
Effect
Model
1.
Freeze Drying
CMC/ZnnHAp/ascorbic acid scaffold Chitosantricalcium phosphatefucoidan scaffold Chitosan– gelatin–siloxane
miR-15b
Promoted osteoblast differentiation
Mouse MSCs (mMSCs)
-
Supported osteogenic differentiation
-
Silk scaffold
Coated with decellularized pulp/collagen/ fibronectin
PCL/PLGA/HA
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No.
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Graphene oxide and nHAp
Enhanced ALP and the expression of bonespecific genes Enhanced biofunctionality and used in bone resorption treatment
Rabbit AD-MSCs
84
Human osteoblastic cells (MG-63)
85
Increased
Bone
86
expression of the Runx2, OPN, OCN, BMP-2, collagen I Increased expression of Runx2, ALP, collagen type 1 and osteocalcin genes Increased cell proliferation, ALP and osteogenic gene expression Promoted osteoblast differentiation
marrow-derived MSCs
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83
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-
-
Alg/Gel/chitosan nanoparticles
Silibinin
Graphene oxide/HAp/silk fibroin scaffold
-
Biphasic calcium phosphate nanoparticles/ collagen composite
Dexamethasone
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Human bone marrow stromal cell
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Gelatin/ CMC cellulose/ PVA/nHAp
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Ref 82
Human bone marrow derived MSCs
87
Rat bone marrowderived MSCs
88
mMSCs
34
Stimulated expression of osteocalcin, and enhanced osteoblast differentiation Promoted osteogenic differentiation
MC3T3-E1
89
Human MSCs
90
Upregulated the expression of ALP, Runx2, IBSP and BMP-2
Athymic nude mouse
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Increased cell proliferation and osteogenic differentiation Enhanced cell proliferation and regeneration capacity
Human bone marrow-derived MSCs
91
MC3T3-E1
92
-
Favored cell attachment, proliferation and differentiation
MC3T3-E1
93
-
Induced new bone formation Accelerated osteogenesis
Graphene oxide/Chitosan/ hyaluronic acid HA/gelatin/PVA
Simvastatin
Chitosan/nHAp/n ZrO2 Chitosan/CaPP
miR-590-5p
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Raloxifene
94
Facilitated growth of new bone Promoted osteoblast differentiation Enhanced osteoblast differentiation at the cellular and molecular levels
Rat tibia
96
mMSCs
77
Mouse MSCs (C3H10T1/2)
97
Increased bone mass density
Wistar rats
Chrysin
Showed proliferative effect and promoted bone growth at cellular and molecular levels
mMSCs
98
Collagen
Promoted osteoblast proliferation
Human fetal Osteoblasts
99
Chitosan/nHAp
Genipin
Murine 7F2 osteoblast-like cells
100
PLLACL/ collagen
Dexamethasone and BMP-2
Promoted osteoblast differentiation and proliferation Showed osteogenic differentiation
Human MSCs
101
PLLA/PCL/HA
-
Enhanced osteoblast differentiation and proliferation
MC3T3-E1
102
PLA/Tussah silk fibroin
-
Enhanced cell adhesion and
mMSCs
103
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-
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hDPCs10/ hPDLCs11 Human adipose derived stem cells
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Pigeonite
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Chitosan/CMC/ nHAp
Electrospinning
PLLA/HA
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2.
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Strontium HAp/ chitosan nanohybrid scaffold Collagen/chitosa n/β-tricalcium phosphate composite loaded with PLGA Alginate/ cellulose nanocrystals/ chitosan/ gelatin composite scaffold Silk fibers/HAp
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95
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Catecholamines and Ca2+
Polyhydroxybutyrate
nHAp
New Zealand rabbit Human fetal osteoblasts
osteoid tissue formation
BALB/c nude mice
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BMP-2
HA/TCP
-
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Fibrin
Improved cell proliferation and osteogenic differentiation Showed osteogenic differentiation
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Bioactive glass
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3D-bioprinting
PLLA
Increased bone density
Rabbit MSCs 105
Human bone marrow-derived MSCs
106
Muscle derived stem cells from mouse and C2C12 Lewis rats
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108
-
Increased material resorption and improved bone formation
Rabbits
109
-
Showed proliferative and osteoblastic differentiation effect
Human fetal osteoblasts
110
H / β-TCP
-
Promoted proliferation and osteoclastic differentiation
RAW 264.7
111
Mesoporous bioactive Glass with PVA
Dexamethasone
Showed osteogenic differentiation and anti-inflammatory properties
hBMSCs
112
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monolithic monetite blocks
PLGA/PGA
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3.
104
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Collagen
Formation of new bone Enhanced cell adhesion, proliferation, differentiation and osteogenic expression of osteocalcin, osteopontin Exhibited better adherence, proliferation and osteogenic phenotype
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ACCEPTED MANUSCRIPT H and β-TCP
Recombinant BMP-2
Possessed proliferative and osteogenic differentiation effect
Mesoporous bioactive glass with alginate
Dexamethasone
Showed osteogenic differentiation property
β-TCP scaffolds of 500,750 and 1000 µM pore sizes
-
Showed proliferation and new bone formation
Hyaluronic acid/PEG
-
β-TCP scaffolds
SrO and MgO
Mouse muscle myoblastic cell line (C2C12) and rabbit dorsal muscle tissue
113
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114
115
Improved bone formation compared to β-TCP
Sprague-Dawley rats Rabbits
117,118
Showed new bone growth
C3H10T1/2 BALB/cJ mice
119
Possessed proliferative and differentiation properties
Human bone marrow-derived MSCs
120
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human fetal osteoblast cells (hFOB) Sprague–Dawley rats Human bone marrow-derived MSCs, mouse endothelial cells (MS1), mouse fibroblast cells (L929)
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Increased expression of CD31, CD105(actin expression) Increased cell-cell interaction, invasion and migration
Collagen
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HAp and alphatricalcium phosphate
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Fe3O4 nanoparticles/ MBG/PCL
Human bone marrow-derived MSCs
116
PEG dimethacrylate/ gelatin methacrylte
-
Showed osteogenic differentiation effect
Human bone marrow-derived MSCs
121
PCL/PLGA/TCP
ECM laid by nasal inferior turbinate tissue
Increased osteogenesis and its markers including Runx2
Human bone marrow-derived MSCs
122
43
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Fibrinogen Pluronic F-127
Increased proliferation and differentiation
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CE
PT
ED
M
AN
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PCL/Gelatin and HAp hydrogels
Calvarial and subcutaneous implantation in Adult Sprague Dawley rats Human amniotic fluid derived stem cells
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123
Figure 1
Figure 2
S. Preethi Soundarya, A. Haritha Menon, S. Viji Chandran, N. Selvamurugan PII: DOI: Reference:
S0141-8130(18)33716-4 doi:10.1016/j.ijbiomac.2018.08.056 BIOMAC 10300
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19 July 2018 7 August 2018 10 August 2018
Please cite this article as: S. Preethi Soundarya, A. Haritha Menon, S. Viji Chandran, N. Selvamurugan , Bone tissue engineering: Scaffold preparation using chitosan and other biomaterials with different design and fabrication techniques. Biomac (2018), doi:10.1016/j.ijbiomac.2018.08.056
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ACCEPTED MANUSCRIPT Bone tissue engineering: Scaffold preparation using chitosan and other biomaterials with different design and fabrication techniques
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S. Preethi Soundarya , A. Haritha Menon , S. Viji Chandran+ and N. Selvamurugan*
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Department of Biotechnology, School of Bioengineering, SRM Institute of Science and
These authors contributed equally to the paper
To whom correspondence should be made:
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*
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+
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Technology, Kattankulathur 603 203, Tamil Nadu, India
Professor
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N. Selvamurugan, Ph.D.
Department of Biotechnology School of Bioengineering SRM Institute of Science and Technology Kattankulathur 603 203. Tamil Nadu India. Cell: 91-9940632335 Email: [email protected] [email protected] 1
ACCEPTED MANUSCRIPT Abstract In the recent years, a paradigm shift is taking place where metallic/synthetic implants and tissue grafts are being replaced by tissue engineering approach. A well designed threedimensional scaffold is one of the fundamental tools to guide tissue formation in vitro and in vivo. Bone is a highly dynamic and an integrative tissue, and thus enormous efforts have been
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invested in bone tissue engineering to design a highly porous scaffold which plays a critical role
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in guiding bone growth and regeneration. Numerous techniques have been developed to
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fabricate highly interconnected, porous scaffold for bone tissue engineering applications with the help of biomolecules such as chitosan, collagen, gelatin, silk, etc. We aim, in this review, to provide an overview of different types of fabrication techniques for scaffold preparation in bone
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tissue engineering using biological macromolecules.
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Keywords: Fabrication, scaffold, bone tissue engineering, chitosan, Runx2
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ACCEPTED MANUSCRIPT Abbreviations Alg - Alginate ALP - Alkaline phosphatase BTE – Bone tissue engineering
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CI - Chitin
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Col - Collagen CMC – Carboxymethyl cellulose
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CS - Chitosan
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FDM - Fused deposited modeling Gel - Gelatin
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HA - Hyaluronic Acid
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HAp – Hydroxyapatite
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LOM - Laminated object manufacturing MSC – Mesenchymal stem cell
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OPN – Osteopontin
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PCL - Polycaprolactone
PEG – Polyethylene glycol PGA - Polyglycolic acid PLGA - Poly-dl-lactic-co-glycolic acid PLLA - Poly-l-lactic acid PVA – Polyvinyl alcohol
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ACCEPTED MANUSCRIPT PS - Polystyrene PVP - Polyvinylpyrrolidone
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Runx2 - Runt-related transcription factor 2
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ACCEPTED MANUSCRIPT Contents 1. Bone-----------------------------------------------------------------------------2. Bone defects---------------------------------------------------------------------3. Bone tissue engineering--------------------------------------------------------4. Biomaterials in bone tissue engineering -------------------------------------4.1 Natural Polymers-------------------------------------------------------
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4.1.1 Chitin/Chitosan ---------------------------------------------
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4.1.2 Collagen/Gelatin---------------------------------------------
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4.1.3 Alginate ------------------------------------------------------4.1.4 Hyaluronic Acid ---------------------------------------------
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4.1.5 Silk ------------------------------------------------------------4.2 Synthetic Polymers------------------------------------------------------
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4.3 Ceramics ---------------------------------------------------------------5. Design of natural composites for bone regeneration ---------------------
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5.1 Biocompatibility property-----------------------------------------------5.2 Biodegradability ----------------------------------------------------------
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5.3 Scaffold architecture ----------------------------------------------------5.4 Mechanical property -----------------------------------------------------
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5.5 Fabrication techniques---------------------------------------------------5.5.1 Freeze drying --------------------------------------------------------
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5.5.2 Solvent casting -----------------------------------------------------5.5.3 Particulate leaching -------------------------------------------------
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5.5.4 Gas foaming --------------------------------------------------------5.5.5 Thermally induced phase separation----------------------------5.5.6 Sol-Gel method----------------------------------------------------5.5.7 Electrospinning ----------------------------------------------------5.5.8 3D-printing ---------------------------------------------------------6. Conclusion----------------------------------------------------------------------7. Future perspective----------------------------------------------------------------
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ACCEPTED MANUSCRIPT 1. Bone Bone is a large hard connective tissue that supports and protects various internal organs and provides structural integrity to the body [1-3]. It undergoes continuous remodeling during the lifetime of an individual and acts as the calcium reservoir of the body [4, 5]. Bone is constituted by collagen which primarily forms the organic part of the bone and calcium phosphate (apatite) crystals that are found as embodiments within the collagen matrix and form
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the inorganic part of the bone [4,6]. Bone mainly comprises of four types of cells namely
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osteoblasts, osteoclasts, osteocytes, and bone lining cells and they dynamically regulate bone
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homeostasis. Bone remodeling is a function of bone formation performed by the osteoblasts, and resorption by osteoclasts and equilibrium between these two functions is important for healing,
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bone regeneration and to maintain the structural integrity of the tissue [7-9]. In mammals, most bone develops through endochondral ossification. In the remodelling process, mesenchymal
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stem cells (MSCs) first differentiate into hypertrophic cartilage followed by differentiation of MSCs into osteoblasts in the surrounding perichondrium that form the initial bone matrix
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followed by vascularization. As this process continues, osteoclasts invade and erode the cartilage to replace the chondrocytes with developed bone tissue [10-12]. The osteoblasts continue to lay
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down the bone matrix to form the mature bone tissue, and while doing so, some osteoblasts deposit onto the matrix, and these form the mature bone cells called osteocytes.
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2. Bone defects
Loss of bone tissue can occur as a consequence of trauma due to accidents, cancer or
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bone defects such as osteoporosis, osteogenesis imperfecta and congenital pseudarthrosis [1214]. In case of long-bone injuries, a significant loss in bone tissue results when the natural
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healing potential of the bone falls far below the required regeneration. Hormonal imbalance caused as a result of aging is also a significant cause of bone loss. Studies showed that the hormone estrogen suppresses osteoclast activation and parathyroid hormone (PTH) regulates bone remodeling [15-18]. Diabetes is yet another major disorder that contributes sizably to the lossof bone tissue. This occurs as a result of elevated levels of free radicals in the surrounding tissues causing an acidic environment, thereby activating the osteoclasts that resorb the bone matrix [19-22]. Allografts and auto-grafts are being employed for replacement of lost bone tissue. However, these options pose major disadvantages such as lack of donors and immunogenic rejection of the graft by the host’s body [23, 24]. To overcome these 6
ACCEPTED MANUSCRIPT disadvantages, bone tissue engineering (BTE) is now being employed in the treatment of bone defect/loss. 3. Bone tissue engineering Creating tissue constructs that mimic the bone in both structure and function has been a challenge so far. A bone graft or scaffold should mimic the structure and properties of the natural bone extracellular matrix (ECM) and provides all the necessary environmental cues
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found in natural bone. This is where BTE comes into play, and the current trends of regenerative
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medicine have focused on the creation of 3D-scaffold with the help of biomaterials and cells that
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can mimic the ECM, support the formation of new tissue/bone, and meanwhile, degrade as new bone is produced [25]. Various technologies come together to construct porous scaffold to
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regenerate tissues/organs and also for the controlled and targeted release of bioactive agents in tissue engineering applications. The optimal characteristics of a scaffold are the strength, the rate
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of degradation, porosity, and microstructure, as well as their shapes and sizes of the polymeric scaffold [26, 27].
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The following are some of the features that need to be possessed by the scaffold to be used in BTE efficiently. (i) The scaffold biomaterial should possess flexibility, i.e. it should be
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easy to fabricate the material into 3D-structures. (ii) The topography of the biomaterial fabricated into 3D-dimensional structures should promote cell adhesion, cell infiltration, and
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growth. This means that these 3D-structures should possess well interconnected pores that allow cell infiltration and nutrient exchange. Any biomaterial that supports attachment of cells and
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growth of bone on its surface is considered to be osteoconductive. (iii) The rate of degradation of the 3D-structured biomaterial is also a very crucial characteristic. The biomaterial must undergo
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resorption as new bone is formed, i.e. the rate of formation of the new bone tissue should be proportional to the rate of degradation of the biomaterial, and its degradation byproducts must not cause inflammation or toxicity in surrounding tissues or the whole system of the host. (iv) Presence of concavities or spaces between individual particles of the biocomposite scaffold, which aid in heterotopic bone formation without being disturbed by high circulation body fluid or mechanical forces due to implant movement [12, 28-30]. 4. Biomaterials in bone tissue engineering Numerous scaffolds are created from a diverse range of biomaterials and were produced using a plethora of fabrication techniques in attempts to regenerate bone in the body. Regardless 7
ACCEPTED MANUSCRIPT of the site of implantation, a number of key factors are to be taken into consideration when designing or determining the suitability of a scaffold for use in tissue engineering. Biomaterials play a crucial role in the field of BTE by acting as artificial frameworks namely scaffolds, matrices, or constructs [31]. Three distinct groups of biomaterials namely natural, synthetic polymers and ceramics are used in the fabrication of scaffold for tissue engineering. Polymers include natural and synthetic polymers [32].
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4.1 Natural Polymers
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Naturally derived polymer such as chitin (CI), chitosan (CS), collagen (Col), gelatin
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(Gel), alginate (Alg), hyaluronic acid (HA) and its composites are resourceful biomaterials that play a predominant role in BTE.
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4.1.1 Chitin/Chitosan
CI is the most abundant natural amino polysaccharide and it is mainly extracted from
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crustaceans (shrimps, crabs, lobsters, etc). CI and CS are considered as a versatile biomaterial. Although CI possesses various industrial applications, its usage is limited in BTE due to its weak
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solubility. On the other hand, the deacetylated CI derivative-CS is a well-established natural polymer. It mainly comprises of units namely (1-4) glycosidic bonds linked d-glucoasmine
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residues with a variable number of randomly located N-acetyl-d-glucosamine (NAG) groups. CS possesses structural resemblance with glycosaminoglycans (GAG), one of the components of
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ECM that interacts with collagen fibers and plays a vital role in cell-cell adhesion. CS has been given great importance in the field of scaffold fabrication as it contains various properties such
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as biocompatible, biodegradable, anti-bacterial effect and many more [31-33]. To enhance the properties of CS such as mechanical strength and structural integrity for BTE applications, other
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natural/synthetic polymers, metals and ceramics are added to CS, and used as 3D-lyophilized scaffolds, hydrogels/film, and electrospun mats. In many studies, polymers such as Alg, Gel, PCL and bioactive nano ceramics such as HAp, SiO2, TiO2, ZrO2, etc have been used to increase the mechanical strength of CS biocomposites [4, 31-39]. 4.1.2 Collagen/Gelatin Col is the most abundant and widely distributed protein present in our body. Col types 1, 2, 3, 5 and 9 are known to form fibres out of the 29 different types of collagen that have been identified so far. Structurally, Col is composed of 25 different alpha chains with each alpha chain composed of repeating units of Gly-X-Y. The X and Y residues may be proline or 48
ACCEPTED MANUSCRIPT hydroxyproline. The fibres are the final quaternary structure of the Col protein [40]. In BTE, Col obtained from various sources such as marine sponge and ruminants is widely used in combination with a variety of other materials for fabrication of electrospun fibres, scaffold matrices and hydrogels to aid in targeted delivery, stem cell differentiation and osteogenesis [ 41, 42]. Recombinant human-like Col is being synthesized in large scale using bacteria and yeast and aided in higher fibroblast compatibility and increased bone growth [43, 44]. The properties
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of Col such as high biocompatibility, matrix mimicking and biodegradation make it a suitable
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candidate for BTE. Col is highly biodegradable and may degrade off quite quickly when
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implanted in the target site. Col-based scaffolds served as the best carrier for targeted delivery small molecules of antagonists of miR-16, which inhibits Runx2 mRNA (a bone transcription
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factor), thereby promoting osteogenesis [45]. However, Col being a protein is difficult to be fabricated by certain fabrication techniques that involve heat and cross-linking without altering
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its native structure [40]. Gel is a product of collagen denaturation and has the RGD (arginine, glycine, and aspartate) binding sequence that improves cell adhesion. It has been widely used
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separately and as a mixture with many natural polymers including Col, proteins such as fibroin, ceramics such as apatite, and other synthetic and semi synthetic polymers [46-48].
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4.1.3 Alginate
Alg is a natural anionic polymer and it is mainly extracted from brown seaweed. It is
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composed of guluronic acid and mannuronic acid, and possesses properties such as less toxicity, high abundance resources, low cost, gel forming property, biocompatibility and has the potential
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to mimic ECMs. Alg composites are prepared by various cross-linking methods, and it is used in wound healing, tissue engineering and delivery/carriers of drug and other bioactive agents in
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biomedical applications. Several Alg-based biocomposite scaffolds have been reported to possess bone regeneration property. Amongst which Alg/CS composites are the most studied composites that is said to regenerate bone tissue. HAp was blended with Alg in co precipitation method to increase the mechanical strength and these scaffolds have also been reported to possess excellent osteogenic effect [49, 50]. Alg was blended with many other polymers/ceramics such as Col, Gel, nano bioactive glass to increase its mechanical strength, adhesive property and osteogenic differentiation ability [50-53].
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ACCEPTED MANUSCRIPT 4.1.4 Hyaluronic Acid HA is a polysaccharide mainly composed of alternating residues of D-glucuronic acid and N-acetyl glucosamine. It is found in almost all the living organisms, mainly in the connective tissues and load bearing joints, etc. HA is highly viscous, elastic, biocompatible, nonimmunogenic and biodegradable nature makes it an excellent polymer of choice for BTE. It has been shown that HA as such or in mixture with other polymers and ceramics promoted bone
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regeneration [54, 55]. HA is mainly used as an aqueous binder, example in HA/HAp/TCP
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scaffolds where it not only increased osteoconduction but improved clinical handling and
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application [56]. In another study, HA/Gel hydrogels were incorporated into biphasic calcium phosphate (BCP) ceramic to obtain a unique micro- and macroporous structure and acted as
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novel bone substitute for BTE [57]. HA-coated scaffolds were also prepared to enhance the bioactivity of scaffolds prepared from synthetic polymers such as PLA, PVP, PLGA, etc [58,
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59]. Therefore, HA offers great potential as a scaffolding material. 4.1.5 Silk
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Silk fibers are produced by silkworms, bees, spiders, mites, and scorpions. They are composed of two different proteins namely silk fibroin (fibrous) and sericin (globular). Silk
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fibroin is structurally composed of heavy chains and light chains. Sericin coats these two chains of fibroin. Silk fibers are known for their elasticity and other strong mechanical properties. Silk
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protein from tasar silk is rich in RGD sequence which makes it a suitable candidate for BTE [60, 61]. MSCs cultured on silk fibers showed an increase in alkaline phosphatase (ALP) activity and
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after 12 days of treatment, they showed increase in mineralization [62]. There are reports indicating that silk fibroin used in combination with other biomaterials showed enhanced bone
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regeneration. Electrosprayed nanoparticles and hydrogels made of silk fibroin and HAp showed increase in cell proliferation, ALP activity, mineralization and osteocalcin expression, thus proving it supports for mature bone formation [63, 64]. Addition of magnetite particles to silk fibroin/ CS scaffolds aided in synthesis of scaffolds with desired pore sizes [65]. Polymers such as carboxyl methyl cellulose in combination with silk fibroin have been used in synthesis of layer-by-layer assembled fibers to form a 3D-scaffold for BTE [66]. Recently, a patented study showed that surface modification of silk fibroin with ethylene glycol increased adhesion of cells to its surface and decreased thrombosis [67]. This application may be employed to enhance the bone regeneration properties of silk fibroin. 10
ACCEPTED MANUSCRIPT 4.2 Synthetic Polymers Numerous synthetic polymers have been used in the attempt to produce scaffold including
polystyrene
(PS),
poly-l-lactic
acid
(PLLA),
polyglycolic
acid
(PGA),
polycaprolactone (PCL), polyvinylpyrrolidone (PVP) and poly-dl-lactic-co-glycolic acid (PLGA). These polymers are often cheaper than natural polymers and also have much success as they can be fabricated with a tailored architecture, long shelf life and production in large scale.
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Their degradation characteristics can be controlled by varying the polymer itself or the
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composition of the individual polymer. They also have drawbacks including the risk of rejection
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due to reduced bioactivity [2, 26, 68-70]. 4.3 Ceramics
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Ceramics such as hydroxyapatite (HA), tricalcium phosphate (TCP), bioactive glasses and glass-ceramics have been shown to have osteoconductivity, high compressive strength, and
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good bone integration. However, their applications in tissue engineering have been limited because of their brittleness, difficulty of shaping for implantation and biodegradability. We can
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overcome these issues by merging natural biomolecules with synthetic polymers, ceramics or by using biocomposite materials that improve scaffold properties, improve tissue interaction and
applications [2, 26, 68-70].
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thereby allow controlled degradation and improve the biocompatibility in tissue engineering
5.1 Biocompatibility
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5. Design of natural composites for bone regeneration
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An important criterion of any scaffold for tissue engineering is that it must be biocompatible; the scaffold must promote cell adhesion, vascularization, the supply of nutrients
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and oxygen to the cells and also support normal cellular activity including molecular signaling systems. After implantation, the scaffold or tissue engineered construct should not elicit an undesirable local or systemic responses to the host. Cytotoxicity, genotoxicity, immunogenicity, mutagenicity, and thrombogenicity are some of the undesirable effects to be eliminated as it might cause severe inflammatory response thus reduce healing or cause rejection by the body [25-26, 71-73]. 5.2 Biodegradability The degradation rate of the scaffold must be in designed in such a way that it provides the necessary structural support and also allows the body's cells, over time, to eventually replace 11
ACCEPTED MANUSCRIPT the implanted scaffold or tissue engineered construct. The scaffold must be biodegradable and are not intended as permanent implants. It is related to biocompatibility because the degraded products should also be non-toxic and able to exit the body without interference with other organs [4, 26, 74-75] 5.3 Scaffold architecture Another indispensable requisite for scaffold is its architecture, porosity and pore size.
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The scaffold should have an interconnected structure and high porosity to ensure cellular
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penetration and adequate diffusion of nutrients to cells within the construct. Cells primarily
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interact with scaffold via the chemical groups or ligands on the material surface. Scaffold synthesized from natural extracellular materials possess these ligands in the form of Arg-Gly-
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Asp (RGD) binding sequences, whereas scaffold made from synthetic materials may require deliberate incorporation of these ligands through, for example, protein adsorption. The ligand
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density is influenced by the available surface within a pore of the scaffold to which cells can adhere [26, 74, 76-77].
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Figure 1 5.4 Mechanical property
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Ideally, the mechanical properties of scaffold should be consistent with the anatomical site into which it is to be implanted, and it must also be strong enough to allow surgical handling
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during implantation. Bone supports the body weight, and it possesses very high compressive strength. In general, the compressive modulus ranges for trabecular bone and cortical bone from
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0.01 to 2.0 GPa and from 14 to 18 GPa, respectively. The scaffold should maintain sufficient mechanical properties until newly formed bone can assume a structured role. Fabricating
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scaffold with passable mechanical properties is one of the great challenges in engineering bone or cartilage. For these tissues, the implanted scaffold should have sufficient mechanical integrity to function from the beginning of implantation to the completion of the remodeling process. A further challenge is that healing rates vary with age; for example, in young individuals, fractures normally heal completely back to its same integrity, but in the elderly, the rate of repair slows down. So this parameter should also be considered when designing a scaffold for orthopedic applications. From this, it is evident that a balance between mechanical properties and porous architecture is key to the success of any scaffold [4, 26, 30, 72, 81,82].
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ACCEPTED MANUSCRIPT 5.5 Fabrication techniques The fabrication technique is the final criterion for designing scaffold for tissue engineering applications and all the criteria listed above depend on it. Table 1 5.5.1 Freeze-drying Lyophilization or freeze-drying has evolved as a conventional fabrication technique for
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BTE. This technique has often been used for food products, biological materials, and drug
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delivery systems. The advantage of this technique is to fabricate a scaffold without the use of a
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high temperature or a separate leaching step and can produce a scaffold of various sizes and shape as required. It is a dehydration technique based on the removal of water/solvent from the
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frozen solution by sublimation under vacuum, at low pressure, leading to an anhydrous or almost anhydrous 3D-structure. The method consists of creating an emulsion by homogenization of a
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polymer solution (in an organic solvent) and water mixture, rapidly cooling the emulsion to lock in the liquid state structure, and removing the solvent and water by freeze-drying. Over 90% of
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porosity can be expected from this technique with the pore size ranging from 20 to 200 µm. The pore size is mostly controlled by controlling the parameters such as rate of the freeze, pH,
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polymer concentration and temperature. For this process, vacuum with high power is needed to come out with a scaffold which has high porosity and interconnectivity. Several natural
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biomolecules and synthetic polymers such as silk proteins, CS, Col, cellulose, Gel, PGA, PLLA, PLGA, PLGA/PPF blends, etc. are widely been used in the freeze-drying technique. But this
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technique is a slow and expensive method, since the cycle may be long, necessitating considerable energy consumption. Freeze-drying can be coupled with techniques such as solvent
124-128].
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casting, particulate leaching, gas foaming, porogen leaching or by emulsion freeze-drying [4, 81,
5.5.2 Solvent casting
It is one of the easiest and inexpensive techniques for fabricating scaffold. It is completely based on the evaporation of the solvent present in the polymeric solution producing porous scaffolds. The scaffold can be prepared by two methods namely (i) The polymer solution is poured into the mold and allowed to rest for the evaporation of the solvent to obtain a porous 3D-scaffold. (ii) The mold is dipped into the polymeric solution and allowed to rest to obtain a polymeric membrane. Some of the solvents used for casting are methylene chloride, methanol, 13
ACCEPTED MANUSCRIPT acetone, dichloromethane, chloroform, etc. The major drawback of this technique is that the solvent used may be toxic and might retain some toxicity thus they are either combined with freeze-drying or particulate leaching technique for better results [129-131]. 5.5.3 Particulate leaching This method involves mixing water-soluble salt (e.g., sodium chloride, sodium citrate), sugar particles or a porogen into a biodegradable polymer solution. The polymeric solution is
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then cast into the mold of the desired shape. After the solvent is removed by lyophilization, the
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salt particles/porogen is leached out to obtain a porous structure. This method is highly
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advantageous because size of the pores can be controlled by salt/polymer ratio and also the size of the particle added. However, the pore shape is limited to the crystal shape of the salt. Another
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drawback is the difficulty in removing soluble salt/particles from the interior of a polymer matrix which makes it hard to fabricate very thick scaffolds. In fact, most of the porous materials
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prepared by solvent casting and particulate leaching method are restricted by thickness [128, 130, 132].
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5.5.4 Gas foaming
Gas foaming process can be used to fabricate highly porous polymer foams without the
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use of any solvents. In this method, an inert gas usually carbon dioxide (CO2) is used as an agent for the formation of polymer foam. Solid polymer disks are exposed to high-pressure gas which
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leads to the nucleation and growth of gas bubbles in the material. They are then freeze-dried to get a 3D-structure with a pore size of around 100 µm and porosity up to 93%. The disadvantage
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of this method is that it mostly yields a closed-pore structure, with less interconnectivity. This can further be combined with other methods to get a better scaffold architecture [128, 133, 134].
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5.5.5 Thermally induced phase separation Thermally induced phase separation technique is based on changes in thermal energy to induce separation of a homogeneous polymer solution into a multi-phase system domain by a quench route. Firstly the polymer is dissolved in a solvent (phenol or naphthalene) at a high temperature, followed by dispersion of biologically active molecule in these solutions. On declining the temperature the separation is induced, the homogeneous solution separates into a polymer-rich phase and solvent-rich phase either by solid-liquid de-mixing or liquid-liquid phase separation mechanism. Then the solvent is removed by extraction, evaporation, and sublimation to give porous scaffold with bioactive molecules integrated into to that structure. The factors that 14
ACCEPTED MANUSCRIPT affect the pore morphology of the scaffold are the type of polymer, solvent, the concentration of the polymer solution and phase separation temperature. The advantage of this technique is that it can easily combine with other fabrication technology and have good mechanical properties to design three-dimensional structures with controlled pore morphology [128, 135]. 5.5.6 Sol-Gel method Simply put, sol-gels are very porous, highly ordered structures analogous to sponges.
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This gel is made up of discrete particles or polymers. Most frequently used precursors are
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chlorides or metal alkoxides. These precursors are hydrolyzed and polycondensed for the
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formation of colloids. Lesser amounts of dopants like organic dyes and also rare earth elements can be used in the sol for homogeneous distribution of the contents present in the final product.
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The major steps involved in the sol-gel process are mixing, casting and gelation. In the stage of mixing, a colloidal solution is formed via mechanical mixing of colloidal particles in water as a
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solvent. The metal alkoxide precursor reacts with water and undergoes hydrolysis and polycondensation reactions. The formation of a colloidal dispersion of extremely small particles
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(1-2 nm) takes place that finally converts into a 3D-network of the corresponding inorganic oxide. In the stage of casting, sol has a low viscosity which makes it easier for it to cast into a
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mold or shape. A suitable mold is chosen to avoid the sticking of the polymeric gel to the mold. During gelation process, 3D-networks start forming from colloidal particles. Gelation results in
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agglomeration with a colloidal particle which is due to interactions among the components which can be electrical. The next steps are the aging and drying of the newly created structure,
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further solidifying the material. It is then followed by dehydration or chemical stabilization of the sol-gel to create the ultrastable, porous material. The final step is densification, tempering
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the gel with high heat to solidify the gel. This process has been extensively used in the field of ceramics, glass, and thin-film coatings. Thin metal oxide films can also be produced using this method. The sol-gel process is preferred due to its economic feasibility and the low-temperature process which gives us control over the composition of the product achieved [136-138]. 5.5.7 Electrospinning Electrospinning technique is a unique approach where the scaffold is prepared using electrostatic forces to produce fine fibers from polymer solutions or melts, and the fibers thus produced have a thickness ranging from nanometer to micrometer and a larger surface area than those obtained from conventional spinning processes. More than 200 different polymers and 15
ACCEPTED MANUSCRIPT composites are used in the electrospinning such as silk fibroin, chitosan, gelatin, collagen, etc. and many synthetic polymers (PCL, PVP, PLLA, etc.) are also used in this technique [130]. A high DC voltage in the range of 10-15 kV is necessary to generate the electrospun fibers, and it is conducted at room temperature with atmospheric conditions. Presently, there are two standard electrospinning setups, vertical and horizontal. Chiefly, an electrospinning set up consists of three major components: a high voltage power supply, a spinneret (e.g., a pipette tip) and a
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grounded collecting plate (usually a metal screen, plate, or rotating mandrel). Most of the
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polymers are dissolved in a solvent to form a homogeneous solution. Then the polymer fluid is
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introduced into the capillary tube and in the presence of a high voltage source, a certain polarity is introduced into the polymer solution or melt, which is then accelerated towards a collector of
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opposite polarity at a constant flow rate [139-140]. However, some polymers may emit an unpleasant or even harmful smell, so the processes should be conducted within chambers having
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a ventilation system [140]. In the electrospinning process, the polymer solution becomes uniformly charged due to the presence of high voltage, and this causes a repulsive force within
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it. When the electric field applied reaches a critical value, the repulsive electrical forces overcome the surface tension forces thereby causing a charged jet of the solution to eject from
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the tip of the Taylor cone and moves towards the opposite polarity or neutral (grounded) charged electrode which is placed nearby that attracts and collects the fibers. The end product is the
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formation of continuous nanofibers. Major advantages of this technique are that it is very versatile, non- invasive and does not require high temperature for the generation of fibers. This
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technique has seen a remarkable increase in research and commercial attention in various fields over the past decade as it can provide structures that are essential for cell growth and subsequent
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tissue organization. With the expansion of this technology, several research groups have developed more sophisticated systems that can fabricate more complex nanofibrous structures in a more controlled and efficient manner [90,91]. These innovations include coaxial electrospinning – the transformation of nanofibers from a single component to complex multilevel structures by multiple feed system through coaxial capillaries, multi-jet, and blow-assisted multi-jet electrospinning technique increased the throughput of the fiber yield and the mutual electrostatic repulsion was reduced in the later technique [141, 142] Many parameters affect the morphology and diameter of electrospun fibers,and some of these important parameters are (i) the type of polymers used, the viscosity of polymer solution, 16
ACCEPTED MANUSCRIPT the surface tension of the solvent in the polymer solution, (ii) the rate of feeding of the polymer solution, the distance between the needle and collector, needle tip size, and (iii) the temperature and humidity of the surrounding environment [143]. Extremely high surface-to-volume ratio, tunable porosity, malleability to conform to a wide variety of sizes and shapes and the ability to control the nanofiber composition are some of the advantages of this technique. The high surface to volume ratio adds benefit as it enables drug loading, cell attachment and infiltration for bone
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growth at the site of implantation [102]. Natural polymers offer good biocompatibility and
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hydrophilicity. However, these polymers often have relatively lesser mechanical strength.
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Therefore, blending synthetic polymers with natural polymers to obtain electrospun fibers overcome these drawbacks alongside decreasing the hydrophobicity of the synthetic constituent
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[144]. Growth factors and phytochemicals have been delivered targeting a specific tissue, and controlled release of these factors could be aided through the blending of different polymers at
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different ratios [139, 145]. 5.5.8 3D-printing
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The traditional methods for fabricating 3D-porous scaffold inBTE applications are such as polyurethane foam templating, solvent casting, melt molding, and freeze-drying; however, it
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is difficult to control the pore interconnection, pore size and overall porosity of the scaffolds. To overcome these, the 3D-printing technique was used to fabricate tissue engineering scaffold
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[120]. In tissue engineering, bioactive cells mixed with hydrogels are deposited layer by layer to form the 3D-structure that mimics the organs [146-150]. These methods create a 3D-scaffold for
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tissue engineering with the help of CAD program like AutoCAD, AutoDesk, etc [151]. 3D-printing, also known as rapid prototyping and additive manufacturing is a technique
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that was first described in the year 1986. This method was initially called stereolithography (SL) by depositing thin layers of a material on top of each other by curing the layers using ultraviolet radiation [152]. Two-photon absorption by photopolymerization for preparation of 3D-organs was derived from this technique where a monomer gel and photo-initiator material were mixed in layers and subjected to the laser. The photo-initiator contains two active photons. On application of the laser, the photons are released and these act as the active species for the monomer to polymerize locally [153, 154]. Similarly, another method was developed namely Selective Laser Sintering (SLS) to make 3D-organs. SLS uses a high power laser to sinter polymer powders to generate a 3D-model [151, 155]. Although it was successful in generating 17
ACCEPTED MANUSCRIPT complex 3D-parts, it had some serious disadvantages. Inventors found that the resolution of printing were direction dependent, i.e. the 3D-parts produced using this method were found to be structurally stronger in one direction but weaker in the other depending on whether the part is parallel to the direction of the laser. The second major disadvantage was the use of high temperatures that caused thermal warpage, and this made the method less suitable to use thermosensitive biological molecules like proteins and cells in the fabrication of 3D-organs [156,
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157].
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Later in the year 2000, the first inkjet-based technique was used for printing proteins and
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endothelial cells into 3D-structures. It was done by modifying the nozzles of a commercial inkjet printer and feeding in software that enabled the user to change the nozzle in the desired x and y
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position. In drug delivery, inkjet printers are most commonly used the technique. Similar to the household inkjet printers, these printing systems deposit small droplets of the drug on liquid
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films that encapsulate the drug droplet to form microparticles which can be used for drug delivery. This method was more advantageous than SLS as no high temperature was employed
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and thermosensitive biomolecules could survive the technique [158, 159]. Powder-based printing utilizes an inkjet printing head that continuously sprays the drug,
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cells or other bioactive molecules along with a binder onto a bed of powdered polymer until a 3D-scaffold is formed. Although this technique aided in the fabrication of scaffold with
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relatively higher mechanical strength, it is not compatible to fabricate 3D-tissue substitutes containing cells or proteins due to the use of high temperature [158, 160, 161]. Many polymers
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such as bentonite, dextrin, maltodextrin, saccharose, edible oils and other resins are used as binders. These binders typically dissolve in the solvent used for bioprinting and upon bringing
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back to room temperature; it hardens and sets to provide mechanical strength to the ceramic/polymer base. Dextrose, maltodextrose,and saccharose are derivatives of sugar and can be easily absorbed as glucose by hydrolysis in our body [161-163]. At very high temperatures, these sugar-based polymers were pyrolyzed to carbon [164, 165]. Polymers such as Teflon (polytetrafluoroethylene) and polypropylene may also be used as binders along with natural or semi-synthetic materials such as collagen as they provide higher mechanical strength to the scaffold and also help in preventing degradation and maintaining the structural integrity of the scaffold while using acidic solvents. However, use of these polymers as binders are disadvantageous as they account for very low porosity and the pore sizes of the scaffold can not 18
ACCEPTED MANUSCRIPT be controlled during synthesis [119]. Acoustic droplet ejection is derived from inkjet printing where an ultrasound pulse ejects low volumes of liquids upwards onto a supporting medium [157, 167-169]. Following these inventions, modifications were done to the existing printers to develop the extrusion-based printing such as Fused deposited modelling (FDM) and Laminated object manufacturing (LOM). Extrusion printing is a modification of inkjet bioprinting where an air
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force plunge or mechanical screw plunger is used to print uninterrupted cylindrical lines rather
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than a single bio-ink droplet [116, 117]. In FDM, a 3D-matrix is fabricated by extruding
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thermoplastic materials and depositing the semi-molten materials onto a stage layer by layer. LOM method uses defined sheets of plastic or metal/plastic composite and stacks them layer by
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layer to form a 3D-scaffold [151, 157, 169]. Extrusion-based printing employs both high and low temperature processes, however at low-temperature, they result in less mechanical strength
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of the scaffolds. FDM is a high-temperature process, and hence only thermoplastic materials are used and are not cytocompatible [169].
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Many 3D-printed polymers and ceramic scaffolds have been tested in vivo for their cytocompatibility and bone regeneration capability. Silicon-substituted HAp/PCL and
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demineralized bone matrix scaffolds prepared using rapid prototyping technique were osteoinductive and showed high osseointegration and aided good bone regeneration in rabbit
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models [170]. Resorbable dicalcium phosphate (DCP) scaffold synthesized using the SLS method was found to enhance bone regeneration in rat models. The synthesis involved the
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conversion of α/β-tricalcium phosphate (Ca3(PO4)2, TCP) powder to brushite (dicalcium phosphate dihydrate) and tricalcium phosphate which upon treatment with phosphoric acid and
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hydrothermal conversion yielded DCP with very high porosity and were not suitable for longterm stable implants [171]. DCP scaffold synthesized by other methods of additive manufacturing such as vat polymerization, powder bed fusion, material extrusion, and binder jetting also showed osteogenic potential in vitro but needs to be validated using in vivo studies [172]. 3D-printed calcium silicate scaffolds, collagen scaffold loaded with DNA complexes and composite calcium phosphate/collagen scaffold implanted into femur defects and PCL/PLGA blended scaffold(using FDM) in the calvarial defects in rats resulted in higher bone regeneration compared to tricalcium phosphate scaffold [119, 173]. Hydrogels are the most preferred biomaterial for tissue regeneration because of their adjustable mechanical properties and 19
ACCEPTED MANUSCRIPT hydrating capability which help it to mimic the biological tissue itself. Cell-laden hydrogels of PEG, gelatin and fibrin and their derivatives have been successfully used in studies to show bone regeneration. Extrusion method is the most common method used for cell-laden hydrogel synthesis [121, 174, 175]. The cell-based printing uses a syringe pump filled with the solvent that moves in z-axis and the substrate moves in y-axis as shown in Fig. 2A [184]. The solvent pump speed and solvent droplet velocity can be controlled by providing differential voltage as
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shown in Fig. 2B[185]. The higher the voltage, the higher would be the solvent droplet velocity.
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This factor is critical, and the velocity of the solvent droplet is to be maintained as it determines
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the pore size and distribution of the scaffold [176, 177]. PCL and PLGA are synthetic polymers, and hydrogels made from these polymers using rapid prototyping showed very good bone
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regeneration property in rabbit tibias [173]. Strikingly, one study used the human-safe filamentous virus that selectively infects bacteria to form a matrix for bone tissue engineering. In
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this study, the RGD sequence was fused to the phage DNA, resulting in several copies of the phage filaments with highly distributed RGD sequences. This was implanted in sites of radial
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bone defects in rats and showed high osteogenesis and angiogenesis in a period of eight weeks [178]. Currently, studies are being done to optimize materials to make a patient-specific scaffold
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for bone regeneration using the computerized tomography (CT) scan of human subjects to print large bone substitutes. Using open source coding software, the CT scans are extrapolated, and
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3D printed to form the bone replacements that are anatomically similar to the scan with a similar pore size and infill density. The scaffold could then be coated or filled with the stem cells from
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the patient and implanted back into the patient’s body, thereby improving bone regeneration and eliminating any chance of rejection [179-183]. Currently, the 3D-printed polymer scaffolds are
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being tested and studied to use as effective drug delivery systems and tissue replacements in the field of surgery and medicine using natural polymers such as CS. Figure 2
6. Conclusion BTE is regarded as the best alternative approach to the conventional bone grafting techniques due to various factors. Scaffold is the basic subunit that provides mechanical strength, a site for cell attachment, proliferation, and differentiation. Polymer selection and scaffold fabrication techniques are the key factors to achieve these goals. The scaffold can be fabricated using a variety of techniques based on the site of implantation, the nature of polymers and their 20
ACCEPTED MANUSCRIPT characteristics. Natural polymers such as CS, Col etc are proved to be excellent for bone regeneration but an ideal fabrication method for successful implantation is yet to be found. A lot of inventions and developments have been made in the last decade in the field of tissue engineering to fabricate an ideal scaffold. Yet, there have been several drawbacks that need to be addressed. Thus, the fabrication of scaffold with advanced techniques using different types of
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biomaterials will still constitute to be the center of research efforts in the field of BTE.
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7. Future perspective
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As discussed, there is a number of a method or techniques used to fabricate a scaffold. While each technique has its own share of merits and demerits, it is necessary to select a suitable method to satisfy the requirements of the specific type of tissue to be repaired. Since the
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requirements of an ideal scaffold for BTE is complex, proficient knowledge from the different fields of science such as biomedical engineering, material science, chemical engineering, etc. is
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required. Extensive process-property optimization will be required to achieve this goal. Demand for novel fabrication technique will increase in the fore coming years due to their ability to
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design a scaffold that can be tailored for a specific patient and clinical needs.
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ACCEPTED MANUSCRIPT Figure legends Figure 1. Macroscopic and microscopic images of scaffolds fabricated by different methods in bone tissue engineering. (A) - (D) show the macroscopic images of scaffolds prepared by freezedrying, electrospinning, sol-gel method, and 3D-bioprinting, respectively, and (E) - (H) show the corresponding SEM images of the scaffolds. Reprinted with permission from [77]
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dental tissue engineering. J. Ind. Eng. Chem. 46 (2017) 175-181. Figure 2. A schematic diagram of a 3D-bioprinter. (A) represents the schematic diagram of a
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3D-bioprinter with the substrate in the moveable Y-axis drive. The Z-axis drive is connected
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to a moveable platform, which is connected to a syringe pump containing the solvent or polymer (B). The syringe pump speed and solvent droplet velocity are controlled by
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supplying differential voltage. Reprinted with permission from [184] T. Peng, Analysis of energy utilization in 3d printing processes. Procedia CIRP. 40 (2016) 62-67; [185] M. Lee H.Y.Kim,
Towardnanoscale
three-dimensional
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electrospunnanofibers. Langmuir. 30(5) (2014) 1210-1214.
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printing:
Nanowalls
built
of
ACCEPTED MANUSCRIPT Table 1. In the study of bone regeneration in vitro and in vivo, a number of the following different types of fabrication techniques, biomaterials, bioactive molecules and experimental model system were used.
Fabrication
Materials
Additive
Effect
Model
1.
Freeze Drying
CMC/ZnnHAp/ascorbic acid scaffold Chitosantricalcium phosphatefucoidan scaffold Chitosan– gelatin–siloxane
miR-15b
Promoted osteoblast differentiation
Mouse MSCs (mMSCs)
-
Supported osteogenic differentiation
-
Silk scaffold
Coated with decellularized pulp/collagen/ fibronectin
PCL/PLGA/HA
-
T
No.
CE
PT
Graphene oxide and nHAp
Enhanced ALP and the expression of bonespecific genes Enhanced biofunctionality and used in bone resorption treatment
Rabbit AD-MSCs
84
Human osteoblastic cells (MG-63)
85
Increased
Bone
86
expression of the Runx2, OPN, OCN, BMP-2, collagen I Increased expression of Runx2, ALP, collagen type 1 and osteocalcin genes Increased cell proliferation, ALP and osteogenic gene expression Promoted osteoblast differentiation
marrow-derived MSCs
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83
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-
-
Alg/Gel/chitosan nanoparticles
Silibinin
Graphene oxide/HAp/silk fibroin scaffold
-
Biphasic calcium phosphate nanoparticles/ collagen composite
Dexamethasone
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Human bone marrow stromal cell
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M ED
Gelatin/ CMC cellulose/ PVA/nHAp
40
Ref 82
Human bone marrow derived MSCs
87
Rat bone marrowderived MSCs
88
mMSCs
34
Stimulated expression of osteocalcin, and enhanced osteoblast differentiation Promoted osteogenic differentiation
MC3T3-E1
89
Human MSCs
90
Upregulated the expression of ALP, Runx2, IBSP and BMP-2
Athymic nude mouse
ACCEPTED MANUSCRIPT -
Increased cell proliferation and osteogenic differentiation Enhanced cell proliferation and regeneration capacity
Human bone marrow-derived MSCs
91
MC3T3-E1
92
-
Favored cell attachment, proliferation and differentiation
MC3T3-E1
93
-
Induced new bone formation Accelerated osteogenesis
Graphene oxide/Chitosan/ hyaluronic acid HA/gelatin/PVA
Simvastatin
Chitosan/nHAp/n ZrO2 Chitosan/CaPP
miR-590-5p
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Raloxifene
94
Facilitated growth of new bone Promoted osteoblast differentiation Enhanced osteoblast differentiation at the cellular and molecular levels
Rat tibia
96
mMSCs
77
Mouse MSCs (C3H10T1/2)
97
Increased bone mass density
Wistar rats
Chrysin
Showed proliferative effect and promoted bone growth at cellular and molecular levels
mMSCs
98
Collagen
Promoted osteoblast proliferation
Human fetal Osteoblasts
99
Chitosan/nHAp
Genipin
Murine 7F2 osteoblast-like cells
100
PLLACL/ collagen
Dexamethasone and BMP-2
Promoted osteoblast differentiation and proliferation Showed osteogenic differentiation
Human MSCs
101
PLLA/PCL/HA
-
Enhanced osteoblast differentiation and proliferation
MC3T3-E1
102
PLA/Tussah silk fibroin
-
Enhanced cell adhesion and
mMSCs
103
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-
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hDPCs10/ hPDLCs11 Human adipose derived stem cells
M
Pigeonite
ED PT
CE
Chitosan/CMC/ nHAp
Electrospinning
PLLA/HA
AC
2.
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Strontium HAp/ chitosan nanohybrid scaffold Collagen/chitosa n/β-tricalcium phosphate composite loaded with PLGA Alginate/ cellulose nanocrystals/ chitosan/ gelatin composite scaffold Silk fibers/HAp
41
95
ACCEPTED MANUSCRIPT proliferation and mineralization
Catecholamines and Ca2+
Polyhydroxybutyrate
nHAp
New Zealand rabbit Human fetal osteoblasts
osteoid tissue formation
BALB/c nude mice
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BMP-2
HA/TCP
-
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Fibrin
Improved cell proliferation and osteogenic differentiation Showed osteogenic differentiation
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Bioactive glass
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3D-bioprinting
PLLA
Increased bone density
Rabbit MSCs 105
Human bone marrow-derived MSCs
106
Muscle derived stem cells from mouse and C2C12 Lewis rats
107
108
-
Increased material resorption and improved bone formation
Rabbits
109
-
Showed proliferative and osteoblastic differentiation effect
Human fetal osteoblasts
110
H / β-TCP
-
Promoted proliferation and osteoclastic differentiation
RAW 264.7
111
Mesoporous bioactive Glass with PVA
Dexamethasone
Showed osteogenic differentiation and anti-inflammatory properties
hBMSCs
112
CE
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monolithic monetite blocks
PLGA/PGA
AC
3.
104
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Collagen
Formation of new bone Enhanced cell adhesion, proliferation, differentiation and osteogenic expression of osteocalcin, osteopontin Exhibited better adherence, proliferation and osteogenic phenotype
42
ACCEPTED MANUSCRIPT H and β-TCP
Recombinant BMP-2
Possessed proliferative and osteogenic differentiation effect
Mesoporous bioactive glass with alginate
Dexamethasone
Showed osteogenic differentiation property
β-TCP scaffolds of 500,750 and 1000 µM pore sizes
-
Showed proliferation and new bone formation
Hyaluronic acid/PEG
-
β-TCP scaffolds
SrO and MgO
Mouse muscle myoblastic cell line (C2C12) and rabbit dorsal muscle tissue
113
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114
115
Improved bone formation compared to β-TCP
Sprague-Dawley rats Rabbits
117,118
Showed new bone growth
C3H10T1/2 BALB/cJ mice
119
Possessed proliferative and differentiation properties
Human bone marrow-derived MSCs
120
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human fetal osteoblast cells (hFOB) Sprague–Dawley rats Human bone marrow-derived MSCs, mouse endothelial cells (MS1), mouse fibroblast cells (L929)
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M
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Increased expression of CD31, CD105(actin expression) Increased cell-cell interaction, invasion and migration
Collagen
CE
HAp and alphatricalcium phosphate
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Fe3O4 nanoparticles/ MBG/PCL
Human bone marrow-derived MSCs
116
PEG dimethacrylate/ gelatin methacrylte
-
Showed osteogenic differentiation effect
Human bone marrow-derived MSCs
121
PCL/PLGA/TCP
ECM laid by nasal inferior turbinate tissue
Increased osteogenesis and its markers including Runx2
Human bone marrow-derived MSCs
122
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ACCEPTED MANUSCRIPT
Fibrinogen Pluronic F-127
Increased proliferation and differentiation
AC
CE
PT
ED
M
AN
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PCL/Gelatin and HAp hydrogels
Calvarial and subcutaneous implantation in Adult Sprague Dawley rats Human amniotic fluid derived stem cells
44
123
Figure 1
Figure 2